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 Caspase酶对应的底物，Caspases（半胱氨酸天冬氨酸蛋白酶，半胱氨酸依赖性天冬氨酸定向蛋白酶）是一类蛋白酶家族，其功能与凋亡（程序性细胞死亡），坏死和发烧（炎症）的过程密切相关。

       什么是胱天蛋白酶？

      胱天蛋白酶（Caspases）是含半胱氨酸的天冬氨酸蛋白水解酶，它们是为细胞凋亡的主要介质。多种受体，例如TNF-α 受体，FasL受体，TLR和死亡受体，以及Bcl-2和凋亡抑制剂（IAP）蛋白家族参与并调节该caspase依赖性凋亡途径。一旦Caspase受到上游信号（外部或内在）刺激被激活，即会参与执行下游蛋白底物的水解作用，并触发一系列事件，导致细胞分解，死亡，吞噬作用和细胞碎片的清除。

      人Caspases酶

      人的Caspases家族基于序列相似性和生物学功能等共性主要可分为三大类：第一类由具有长胱天蛋白酶募集结构域的“炎症”胱天蛋白酶组成，他们对P4位上的较大的芳香族或疏水性残基具有亲和力。第二类由具有短的前体结构域的“细胞凋亡效应”胱天蛋白酶组成，而第三类由具有长的前提结构域的Pap位置具有亮氨酸或缬氨酸底物亲和力的“凋亡引发剂”胱天蛋白酶组成（表1）。

       表1. 人胱天蛋白酶的功能分类：

       启动器Caspase和效应器Caspase酶

      根据其在凋亡胱天蛋白酶途径中的作用，胱天蛋白酶可分为两类：启动器和效应器Caspase酶。启动器和效应器Caspas酶都具有由小亚基和大亚基组成的催化位点，Caspase酶的识别位

      凋亡启动器Caspase酶，例如caspase-2，-8，-9和-10可以启动caspase激活级联反应。Caspase-8对于形成死亡诱导信号复合物（DISC）是必不可少的，并且在激活后，Caspase-8激活下游效应子Caspase（例如Caspase 3）并介导线粒体中细胞色素c的释放。Caspase-8已被证明对IETD肽序列具有相对较高的底物选择性。凋亡效应胱天蛋白酶例如Caspase-3，-6和-7虽然不负责启动级联途径，但是当被激活时，它们在级联的中间和后续步骤中起着不可或缺的作用。Caspase-3（CPP32 / apopain）是关键效应器，因为它放大了来自启动器Caspase的信号，使用对Caspase-3有选择性的DEVD肽序列对活化的Caspase-3进行检测，可以检测Caspase-3的活性。

       Caspase酶底物和抑制剂

      Caspase底物和抑制剂由两个关键成分组成：Caspase识别序列和信号产生或蛋白酶抑制基序。不同Caspase识别序列不同，一般由三个或四个氨基酸组成（表2）。Caspase酶识别序列的N端通常有乙酰基（Ac）或碳苯甲氧基（Z）基团修饰，以增强膜的通透性。对应的Caspase识别特定的肽序列为其酶促反应切割位点，释放产生信号或抑制信号的基序。Caspase的显色和荧光底物均以相似的方式起作用，其中底物的信号或颜色强度与蛋白水解活性成正比。

       表2. Caspase的底物及其序列

         Caspase酶的显色底物

      Caspase的显色底物是有Caspase识别序列及生色基团组成，常见的生色团有pNA（对硝基苯胺或4-硝基苯胺），可使用酶标仪或分光光度计在405 nm处进行光密度检测。

       表3. Caspase的显色底物

       Caspase的荧光底物

      Caspase的荧光底物的结构包含与半胱天冬酶识别相关的荧光团，例如7-氨基-4-甲基香豆素（AMC），7-氨基-4-三氟甲基香豆素（AFC）， Rhodamine 110（R110）或ProRed™620。R110的Caspase底物比基于香豆素的Caspase底物（例如AMC和AFC）更敏感，但由于两步裂解过程，其动态范围更窄。 建议将R110标记的Caspase底物用于终点法测定，而将AMC和AFC标记的 Caspase底物用于动力学测定。

      图.从左到右，分别是AMC（7-氨基-4-甲基香豆素），AFC（7-氨基-4-三氟甲基香豆素），Rhodamine 110（R110）和ProRed™620的激发和发射光谱。

       表4.荧光半胱天冬酶底物。

       注意：

        1.ε=在其最大吸收波长处的摩尔消光系数（单位= cm ^-1M ^-1）。

      2.Φ=水性缓冲液（pH 7.2）中的荧光量子产率。

       Caspase抑制剂

      Caspase抑制剂能与Caspase的活性位点结合并形成可逆或不可逆的连接，通常，Caspase抑制剂的结构由Caspase识别序列，诸如醛（-CHO）或氟甲基酮（-FMK）的官能团组成。具有醛官能团的胱天蛋白酶抑制剂是可逆的，而具有FMK的抑制剂是不可逆的。半胱天冬酶底物和抑制剂都具有较小的细胞毒性作用，因此，它们是研究半胱天冬酶活性的有用工具。

       表5. 可逆和不可逆的Caspase酶抑制剂

Definition

The polypeptide hormone interleukin-1 (IL-1) is one of the key mediators of host's response to microbial invasion, inflammation, tissue injury, or immunological reactions. IL-1 is a prominent member of a group of polypeptide mediators now called cytokines.

Related Peptides

The IL-1 gene family is composed of IL-1a, IL-1b, and IL-1Ra. Each member is first synthesized as a precursor protein; the precursors for IL-1 (prolL-1a and prolL-1b) each have a molecular mass of 31 kDa. The prolL-1a and mature 17 kDa lL-1a are both biologically active. In contrast, prolL-1b is relatively inactive and requires cleavage to a 17-kDa peptide for optimal biological activity. The IL-1Ra precursor has a leader sequence, is cleaved to its mature form, and is secreted like most proteins1.

Structural Characteristics

IL-1 ligands (IL-1a and IL-1b, collectively referred to as IL-1) are pluripotent, proinflammatory cytokines. These two IL l’s share only small stretches of amino acid homology (26% in the case of human IL 1). Each IL 1 is coded by a separate gene, both genes are located on chromosome 2, and each gene contains seven exons. mRNA coding for IL-1b predominates over that coding for IL-1a and this prevalence of IL-1b has been observed in the proportion of the two IL 1 forms measured in the circulation and other body fluids. Both forms of IL 1 are unique in that they are initially translated as precursor polypeptides (31,000 kDa), and despite the fact that IL 1 is found in the extracellular compartment, neither form contains a signal cleavage sequence. The generation of the NH2 terminus of the mature peptide (17,500 kDa) and smaller peptides occurs by the action of senine proteases. Of particular interest is the a/b homologous region termed C-D, which is coded entirely by the sixth exon. It has been suggest that this region may contain the minimal recognition site for IL 1 receptors. Receptors for IL 1 equally recognize the b  and a forms, both forms possess the same spectrum of biological properties, and molecular modeling studies reveal that the two IL l’s are composed of b - folded sheets2,3.

Mode of Action

IL 1 specifically binds to a variety of cells. Studies with T lymphocytes and fibroblasts suggest the existence of a single class of high-affinity receptor with a dissociation constant (KD) that varies from 5 to 50 pM with 100-4000 binding sites per cell. The high-affinity receptors are rapidly internalized and bind to nuclear structures, and responsiveness to IL 1 is down-regulated. The rapid down-regulation of IL 1 receptor is specific and may account, in part, for modulating IL1 effects in several cells4. In cells stimulated with IL1, cytosolic calcium increases, sodium/potassium ion fluxes occur, and protein kinase activity increases4.

Functions

Interleukin-1: Regulator of Neuroinflammation - Interleukins 1a and 1b (IL-1) are very potent signaling molecules that are expressed normally at low levels, but are induced rapidly in response to local or peripheral insults. IL-1 coordinates systemic host defense responses to pathogens and to injury within the central nervous system (CNS). Numerous reports have correlated the presence of IL-1 in the injured or diseased brain, and its effects on neurons and nonneuronal cells in the CNS, it has been recently shown that the importance of IL-1 signaling. Further it has been demonstrate that IL-1 is at or near the top of the hierarchical cytokine signaling cascade in the CNS that results in the activation of endogenous microglia and vascular endothelial cells to recruit peripheral leukocytes (i.e., neuroinflammation). The IL-1 system thus provides an attractive target for therapeutic intervention to ameliorate the destructive consequences of neuroinflammation5.

Role of interleukin-1 in stress responses - Stress responses have been characterized as central neurohormonal changes, as well as behavioral and physiological changes. Administration of IL-1 has been shown to induce effects comparable to stress-induced changes. IL-1 acts on the brain, especially the hypothalamus, to enhance release of monoamines, such as norepinephrine, dopamine, and serotonin, as well as secretion of corticotropin-releasing hormone (CRH). IL-1-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis in vivo depends on secretion of CRH, an intact pituitary, and the ventral noradrenergic bundle that innervates the CRH-containing neurons in the paraventricular nucleus of the hypothalamus. Recent studies have shown that IL-1 is present within neurons in the brain, suggesting that IL-1 functions in neuronal transmission. It has been shown that IL-1 in the brain is involved in the stress response, and that stress-induced activation of monoamine release and the HPA axis were inhibited by IL-1 receptor antagonist (IL-1Ra) administration directly into the rat hypothalamus. IL-1Ra has been known to exert a blocking effect on IL-1 by competitively inhibiting the binding of IL-1 to IL-1 receptors6.

References

1.     Dinarello CA (1994). The interleukin-1 family. 10 years of discovery. FASEBJ, 8(15):1314-25.

2.     Auron PE, Warner SJ, Webb AC, Cannon JG, Bernheim HA, McAdam KJ, Rosenwasser LJ, LoPreste G, Mucci SF, Dinarello CA (1987). Studies on the molecular nature of human interleukin-1. J. Immunol, 138(5):1447-1456.

3.     Dinarello C A (1988). Biology of interleukin 1. FASEBJ, 2: 108-115.

4.     Mizel S B, Kilian P L, Lewis J C, Paganelli KA, Chizzonite RA (1987). The interleukin 1 receptor. Dynamics of interleukin 1 binding and internalization in T cells and fibroblasts. J. Immunol., 138: 2906-2912.

5.     Basu A, Krady JK, Levison SW(2004). Interleukin-1: A Master Regulator of Neuroinflammation. Journal of Neuroscience Research, 78:151–156.

6.     Shintani F, Nakaki T, Kanba S, Kato R, Asai M(1995). Role of interleukin-1 in stress responses - A putative neurotransmitter. Molecular Neurobiology, 10: 47-71.

多肽H2N-Asn-Glu-Ala-Tyr-Val-His-Asp-Ala-Pro-Val-Arg-Ser-Leu-Asn-COOH的合成步骤：

1、合成CTC树脂：称取0.72g CTC Resin（如初始取代度约为0.84mmol/g）和0.73mmol Fmoc-Asn(Trt)-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入1.81mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到  Fmoc-Asn(Trt)-CTC Resin。结构图如下：

2、脱Fmoc：加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Asn(Trt)-CTC Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：

3、缩合：取1.81mmol Fmoc-Leu-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入3.63mmol DIPEA，1.72mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Leu-Asn(Trt)-CTC Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：

4、依次循环步骤二、步骤三，依次得到

H2N-Leu-Asn(Trt)-CTC Resin

Fmoc-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Ala-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Ala-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Glu(OtBu)-Ala-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

H2N-Glu(OtBu)-Ala-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

Fmoc-Asn(Trt)-Glu(OtBu)-Ala-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin

以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。

最后再经过步骤二得到 H2N-Asn(Trt)-Glu(OtBu)-Ala-Tyr(tBu)-Val-His(Trt)-Asp(OtBu)-Ala-Pro-Val-Arg(Pbf)-Ser(tBu)-Leu-Asn(Trt)-CTC Resin，结构如下：

5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品H2N-Asn-Glu-Ala-Tyr-Val-His-Asp-Ala-Pro-Val-Arg-Ser-Leu-Asn-COOH。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）

6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。

7、最后总结：

杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽等。

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